Activator of G-protein Signaling (AGS) proteins provide alternate modes of signal input to heterotrimeric G- proteins and diversify G-protein signaling through unexpected mechanisms. Group II AGS proteins contain 1-4 G-protein regulatory (GPR) motifs that serve as a docking site for Gi1-GDP free of G23. Group II AGS proteins include AGS3 and AGS5/LGN, which play important regulatory roles in cell division, neuronal development, synaptic plasticity, the biology of addiction, energy homeostasis, and cardiovascular functions. A long-term objective of this research effort is to determine the functional role of the Group II AGS protein AGS4 in regulating G-protein signaling with a specific focus on the immune system where AGS4 is enriched in lymphocytes. As the first approach to this longer term objective, this proposal addresses key questions related to the interaction of AGS4 with G-proteins and the impact of this interaction on signal processing in the cell. Specific aim #1 Determine the factors and mechanisms that influence AGS4 interaction with Gi1 subunits to test the hypothesis that the AGS4-Gi1 interaction is a regulated event. Specific aim #2 Determine the factors that influence the subcellular distribution of AGS4 to test the hypothesis that positioning of AGS4 is regulated and influences its interaction with G-protein. Specific aim #3 Determine the effect of altered AGS4 levels on G-protein signaling in S1A and Jurkat T cells to test the hypothesis that AGS4 modulates GPCR - G-protein signal processing. Group II AGS proteins like AGS4 provide insight into novel modes of signal input and regulation of heterotrimeric G-protein signaling and provide a platform for discovering mechanisms underlying signal strength, specificity, and integration of G-protein mediated cellular responses. Furthermore, they provide unexpected targets for development of therapeutics for diseases which result from altered heterotrimeric G-protein signaling. PUBLIC HEALTH RELEVANCE: The work proposed in this application focuses on basic mechanisms for regulation of a major system that controls function of various tissues in the body including the immune system. The area of investigation has merit from the perspective of disease diagnostics and the development of new therapeutic strategies.